The invention concerns a method for obtaining hepatitis-safe, sterile, hemoglobin solutions free of pyrogens and stroma and having a low potassium content, by the treatment of erythrocytes with propiolactone, hemolysis, treatment of the hemolyzate with a cation exchange resin in H.sup.+ form, quantitative separation of the stroma lipid, and sterilization by filtration.
Hemoglobin solutions are capable of transporting oxygen in vivo independently of erythrocytes. In order for such solutions to be physiologically tolerable, however, it is necessary to remove as completely as possible the plasma and cell components of the blood from which the hemoglobin is released by hemolysis (plasma proteins, stroma, leucocytes, thrombocytes).
German Pat. No. 2,248,475 discloses a process for preparing such hemoglobin solutions, in which the separation of plasma proteins and erythrocytes is accomplished by floating the erythrocytes in physiological electrolyte solutions and then separating them by centrifugation in discontinuously operating centrifuges.
In "Appraisal of Hemoglobin Solution as a Blood Substitute" by Frank De Venuto, Harold J. Friedmann, J. Ryan Neville and Carl C. Peck, Surgery, Gynecology & Obstetrics, Sept. 1979, Vol. 149, p. 417-436, different methods are described whereby hemoglobin is obtained by crystallization.
Neither the discontinuous centrifugation (blood centrifuge) that is necessary for the washing of erythrocytes nor the dialysis that is necessary in crystallization processes are suitable procedures for the preparation of such large amounts of hemoglobin solution as would be needed for clinical use. Ordinary discontinuously operating centrifuges have a capacity, for example, of only about six liters. The use of a plurality of discontinuously operating centrifuges would be extremely uneconomical.
Washing erythrocytes with electrolyte solutions results in a high electrolyte content in the end product, which would be a disadvantage in the case of special applications, such as the perfusion of coronary heart vessels (cardioplegia) in open-heart surgery.
With the exception of the process described in German Pat. No. 2,248,475, the transmission of serum hepatitis cannot be entirely prevented in any of the other methods of hemoglobin production. However, even the method of sterilization with .beta.-propiolactone described in German Pat. No. 2,248,475 has the disadvantage that this treatment is the first step in the process, which means that the amount of .beta.-propiolactone used acts on any residual amounts of plasma proteins remaining, depending on the method used in preparing the erythrocyte concentrate. Thus a different .beta.-propiolactone concentration acts on the erythrocytes, depending on the amount of residual plasma.
Continuously operating centrifuges are known for the separation of large amounts of blood into cell components and plasma, and they are used, for example, in slaughterhouses for the recovery of a hemoglobin-free plasma. In this process, however, the erythrocytes are so severely damaged that they are nearly entirely destroyed, especially if they pass repeatedly through the centrifuge. Therefore, this continuous centrifugation is not appropriate for the washing of erythrocytes.